{"gene":"UBE2W","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2011,"finding":"Ube2w acts as an initiator E2 that monoubiquitinates CHIP (the E3 ligase), and this monoubiquitination stabilizes the interaction between CHIP and the deubiquitinase ataxin-3; ataxin-3 then limits polyubiquitin chain length on CHIP substrates and subsequently deubiquitinates CHIP to terminate the ubiquitination cycle.","method":"In vitro ubiquitination assays, co-immunoprecipitation, and functional DUB activity assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and in vitro biochemical assays with multiple orthogonal methods; replicated across functional readouts in a single rigorous study","pmids":["21855799"],"is_preprint":false},{"year":2013,"finding":"Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity; it conjugates ubiquitin to the α-amino group of protein N-termini (including its own N-terminus and that of SUMO) rather than to lysine ε-amino groups.","method":"In vitro ubiquitylation assays, mass spectrometry identification of modification site, and biochemical assays with RNF4","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mass spectrometry site identification, independently replicated in same year by Scaglione et al. (PMID:23696636)","pmids":["23560854","23696636"],"is_preprint":false},{"year":2013,"finding":"Ube2w ubiquitinates the N-terminal α-NH2 group of substrates (not internal lysines); this was demonstrated using lysine-less and N-terminally blocked versions of ataxin-3 and Tau, and confirmed by direct N-terminal sequencing of ubiquitinated ataxin-3; Ube2w contains novel active-site residues important for this activity.","method":"Mass spectrometry, in vitro ubiquitination assays with lysine-less and N-terminally blocked substrates, N-terminal sequencing, active-site mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (MS, mutagenesis, N-terminal sequencing, blocked substrate controls) in a single rigorous study","pmids":["23696636"],"is_preprint":false},{"year":2013,"finding":"UBE2W forms a noncovalent homodimer via residues in the β-sheet region and Loops 4 and 7 of the catalytic domain; mutation of two catalytic domain residues disrupts dimerization, but dimerization is not required for ubiquitin transfer activity; the C-terminal region of UBE2W adopts a noncanonical position distinct from all other known E2 structures.","method":"Biochemical dimerization assays, mutagenesis, structural characterization","journal":"Cell biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis and biochemical assays in a single lab with multiple orthogonal approaches","pmids":["23709311"],"is_preprint":false},{"year":2014,"finding":"Ube2w recognizes the backbone atoms of intrinsically disordered N-termini of substrates (not sequence-specific); its own C-terminus is partly disordered and flexible, and this flexibility is required for substrate binding and modification — point mutations in or removal of the flexible C-terminus inhibits substrate binding and N-terminal ubiquitination.","method":"NMR solution ensemble of full-length Ube2w, mutagenesis, in vitro ubiquitination assays, computational modeling","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with functional mutagenesis validation and in vitro reconstitution in a single rigorous study","pmids":["25436519"],"is_preprint":false},{"year":2015,"finding":"TRIM5α employs Ube2W to anchor Lys63-linked polyubiquitin chains via Ube2W-catalyzed monoubiquitination of TRIM5α (targeting internal lysines 45 and 50, not the N-terminus which is αN-acetylated in cells); these anchored chains are then elongated by Ube2N/Ube2V2; this anchored K63-linked polyUb is required for TRIM5α-mediated restriction of viral reverse transcription.","method":"In vitro ubiquitination assays, cell-based E2 depletion, ubiquitin mutant studies, retroviral restriction assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro reconstitution combined with cell-based depletion and functional antiviral readout, multiple orthogonal methods","pmids":["26101372"],"is_preprint":false},{"year":2010,"finding":"UBE2W interacts with the FANCL E3 ligase via FANCL's PHD domain, catalyzes monoubiquitination of FANCL's PHD domain in vitro, and promotes monoubiquitination of FANCD2 in cells in response to UV irradiation (but not MMC), indicating a DNA-damage pathway-specific regulatory role distinct from UBE2T/HHR6.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, overexpression and knockdown in mammalian cells, domain mapping","journal":"Molecules and cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assay and in vitro ubiquitination with cellular knockdown/overexpression, single lab","pmids":["21229326"],"is_preprint":false},{"year":2015,"finding":"Ube2w KO mice show postnatal lethality and defects in testis, thymus, and skin; proteomic analysis of presymptomatic testis shows preferential accumulation of intrinsically disordered proteins in the absence of UBE2W, supporting the model that UBE2W preferentially targets disordered polypeptides in vivo; UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm.","method":"Germline knockout mouse generation, proteomics/mass spectrometry, immunofluorescence for subcellular localization, histology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean germline KO with multiple phenotypic readouts, proteomics, and localization by direct imaging","pmids":["26601958"],"is_preprint":false},{"year":2016,"finding":"Genetic epistasis in chicken and human cells shows that Ube2w does not have an essential role in the DNA damage response, but that loss of Ube2w suppresses the DNA damage hypersensitivity of Rnf4-deficient cells, indicating that Ube2w and Rnf4 function in distinct pathways in response to DNA damage despite their functional interaction in vitro.","method":"Genetic epistasis (double-mutant/knockdown), DNA damage sensitivity assays in chicken and human cells","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in two cell systems, single lab","pmids":["27185577"],"is_preprint":false},{"year":2017,"finding":"Ube2w deficiency markedly decreases mutant huntingtin (mHTT) aggregate formation and increases soluble monomeric mHTT in cultured cells and in HdhQ200 knock-in mice, while reducing mHTT-induced cytotoxicity; this places Ube2w upstream of mHTT aggregation and implicates N-terminal ubiquitination of the disordered HTT N-terminus in promoting aggregation.","method":"Ube2w knockdown/KO in immortalized cells, primary neurons, and knock-in mouse model; filter trap aggregation assays; cell viability assays","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in multiple model systems with specific aggregation and viability readouts, single lab","pmids":["28986324"],"is_preprint":false},{"year":2021,"finding":"UBE2W N-terminally ubiquitinates UCHL1 and UCHL5 at their N-termini; N-terminal ubiquitination of these deubiquitinases distinctly alters their DUB activity; antibodies selectively recognizing N-terminal diglycine remnants (not isopeptide-linked diglycine on lysine) were used to map endogenous UBE2W substrates by mass spectrometry proteomics.","method":"Monoclonal antibody development, antibody-crystal structure determination, mass spectrometry proteomics, DUB activity assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of antibody-peptide complex, MS proteomics for substrate mapping, and functional DUB activity assays in a single study","pmids":["34326324"],"is_preprint":false},{"year":2023,"finding":"UBE2W acts as a writer of aminoacylated lysine ubiquitination, a newly identified PTM in which ubiquitin is attached to the α-amine of aminoacylated lysine residues; over 2,000 such ubiquitination sites were identified on all 20 aminoacylated lysines in human cell lines, and this modification mediates rapid protein degradation.","method":"Genetic code expansion with unnatural amino acids as chemical probes, mass spectrometry, cell-based ubiquitination assays","journal":"Nature structural & molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic code expansion strategy with MS validation, single lab, novel PTM requiring independent replication","pmids":["36593310"],"is_preprint":false},{"year":2022,"finding":"UBE2W inhibits TNFα-induced NF-κB transcriptional activity by attenuating IκB and p65 phosphorylation and blocking nuclear entry of p65; UBE2W overexpression ameliorates DSS-induced colitis in mice with reduced pro-inflammatory cytokine expression.","method":"Dual-luciferase reporter assay, Western blot, immunofluorescence, mouse colitis model with UBE2W overexpression/knockdown","journal":"Digestive diseases and sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, cell-based reporter and mouse model, no direct mechanistic substrate identified for NF-κB pathway inhibition","pmids":["35314916"],"is_preprint":false},{"year":2026,"finding":"UBE2W mediates K63-linked ubiquitination of p53, regulates p53 subcellular localization, and thereby impairs p53-mediated DNA damage repair and apoptotic pathways in pancreatic cancer cells.","method":"In vitro and in vivo functional experiments, ubiquitination assays, subcellular fractionation, knockdown/overexpression","journal":"American journal of cancer research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, limited methodological detail in abstract, mechanistic claim of K63-linked ubiquitination conflicts with established N-terminal ubiquitination activity of UBE2W and requires independent verification","pmids":["41657785"],"is_preprint":false}],"current_model":"UBE2W is an E2 ubiquitin-conjugating enzyme that uniquely catalyzes monoubiquitination at the α-amino group of protein N-termini, preferentially targeting substrates with intrinsically disordered N-termini by recognizing backbone atoms rather than sequence; this activity is mediated by its own flexible, partly disordered C-terminus, and it functions in diverse cellular processes including initiating CHIP ubiquitination cycles (with ataxin-3 as a regulatory DUB partner), anchoring Lys63-linked polyubiquitin chains on TRIM5α for antiviral restriction, promoting huntingtin aggregation, and regulating FANCD2 monoubiquitination in the DNA damage response."},"narrative":{"mechanistic_narrative":"UBE2W is an E2 ubiquitin-conjugating enzyme that catalyzes a distinctive form of monoubiquitination, conjugating ubiquitin to the α-amino group of protein N-termini rather than to the ε-amino group of internal lysines [PMID:23560854, PMID:23696636]. Substrate selection is structural rather than sequence-specific: UBE2W recognizes the backbone atoms of intrinsically disordered N-termini, and its own flexible, partly disordered C-terminus is required for substrate engagement and N-terminal modification [PMID:25436519]. Consistent with this preference, loss of UBE2W in vivo causes preferential accumulation of intrinsically disordered proteins [PMID:26601958]. Through this activity UBE2W functions as an initiator E2 across several pathways: it monoubiquitinates the E3 ligase CHIP to nucleate a ubiquitination cycle regulated by the deubiquitinase ataxin-3 [PMID:21855799], anchors Lys63-linked polyubiquitin chains on TRIM5α (via internal lysines K45/K50) that are then elongated by Ube2N/Ube2V2 to drive antiviral restriction [PMID:26101372], and N-terminally ubiquitinates the deubiquitinases UCHL1 and UCHL5 to alter their catalytic activity [PMID:34326324]. UBE2W also promotes mutant huntingtin aggregation by acting upstream of aggregate formation [PMID:28986324] and contributes to regulation of FANCD2 monoubiquitination in the UV-damage response [PMID:21229326]. Germline knockout mice display postnatal lethality with defects in testis, thymus, and skin, and UBE2W is a ubiquitously expressed cytoplasmic enzyme [PMID:26601958].","teleology":[{"year":2010,"claim":"Before its catalytic specificity was known, UBE2W was placed in a DNA-damage context by showing it engages the FANCL E3 ligase and modulates FANCD2 monoubiquitination, establishing a pathway-specific role distinct from the canonical UBE2T E2.","evidence":"Co-IP, domain mapping, in vitro ubiquitination and cellular knockdown/overexpression in mammalian cells","pmids":["21229326"],"confidence":"Medium","gaps":["Did not define the chemical nature of the ubiquitin linkage formed","UV-specific but not MMC-specific role left the in vivo relevance unresolved","No structural basis for FANCL recognition"]},{"year":2011,"claim":"Established UBE2W as an initiator E2 that monoubiquitinates the E3 ligase CHIP, defining a regulatory ubiquitination cycle terminated by the DUB ataxin-3.","evidence":"In vitro ubiquitination, reciprocal co-IP, and DUB activity assays","pmids":["21855799"],"confidence":"High","gaps":["Site of CHIP modification not chemically defined at this stage","In vivo significance of the cycle not tested","Did not establish substrate-selection principles"]},{"year":2013,"claim":"Resolved the central mechanistic question of UBE2W chemistry by demonstrating it conjugates ubiquitin to the α-amino group of protein N-termini, not internal lysines, redefining it as a protein N-terminal monoubiquitylation enzyme.","evidence":"In vitro ubiquitylation with lysine-less and N-terminally blocked substrates, mass spectrometry, N-terminal sequencing, and active-site mutagenesis","pmids":["23560854","23696636"],"confidence":"High","gaps":["Did not explain how disordered substrates are recognized","Physiological substrate repertoire unknown","Structural basis of N-terminal targeting unresolved"]},{"year":2013,"claim":"Characterized UBE2W as a noncovalent homodimer with a noncanonical C-terminal position distinct from other E2s, showing dimerization is dispensable for ubiquitin transfer.","evidence":"Biochemical dimerization assays, mutagenesis, and structural characterization","pmids":["23709311"],"confidence":"Medium","gaps":["Functional purpose of dimerization unclear","Did not link the noncanonical C-terminus to substrate recognition"]},{"year":2014,"claim":"Explained substrate selectivity by showing UBE2W recognizes backbone atoms of intrinsically disordered N-termini and that its own flexible C-terminus is required for substrate binding and modification.","evidence":"NMR solution ensemble of full-length Ube2w, mutagenesis, in vitro ubiquitination, and computational modeling","pmids":["25436519"],"confidence":"High","gaps":["Did not enumerate endogenous disordered substrates","Affinity determinants for specific substrates not mapped"]},{"year":2015,"claim":"Demonstrated a defined antiviral function in which UBE2W monoubiquitinates internal lysines of TRIM5α to anchor K63 chains elongated by Ube2N/Ube2V2, required for restriction of viral reverse transcription.","evidence":"In vitro ubiquitination, cell-based E2 depletion, ubiquitin mutant studies, and retroviral restriction assays","pmids":["26101372"],"confidence":"High","gaps":["TRIM5α is targeted at internal lysines (N-terminus α-acetylated), showing context-dependent site selection not fully generalized","Recruitment mechanism to TRIM5α not defined"]},{"year":2015,"claim":"Established the in vivo requirement for UBE2W and validated its disordered-protein preference through a knockout mouse showing postnatal lethality and preferential accumulation of intrinsically disordered proteins.","evidence":"Germline knockout mice, proteomics, immunofluorescence localization, and histology","pmids":["26601958"],"confidence":"High","gaps":["Tissue-specific substrate drivers of phenotypes not identified","Did not connect organismal phenotypes to specific N-terminal ubiquitination events"]},{"year":2016,"claim":"Genetic epistasis clarified that despite an in vitro interaction with RNF4, UBE2W and RNF4 act in distinct DNA-damage pathways, and UBE2W is not essential for the DNA damage response.","evidence":"Genetic epistasis with DNA damage sensitivity assays in chicken and human cells","pmids":["27185577"],"confidence":"Medium","gaps":["Mechanism by which Ube2w loss suppresses Rnf4-deficient hypersensitivity unknown","Relevant substrates in the pathway not identified"]},{"year":2017,"claim":"Linked UBE2W to neurodegeneration by placing it upstream of mutant huntingtin aggregation, implicating N-terminal ubiquitination of the disordered HTT N-terminus in promoting aggregate formation and toxicity.","evidence":"Knockdown/KO in cells, primary neurons, and HdhQ200 knock-in mice; filter trap and viability assays","pmids":["28986324"],"confidence":"Medium","gaps":["Direct N-terminal ubiquitination of HTT not biochemically demonstrated in this study","Whether effect generalizes to other aggregation-prone disordered proteins not tested"]},{"year":2021,"claim":"Enabled endogenous substrate mapping with N-terminal-diGly-specific antibodies and showed UBE2W N-terminally ubiquitinates the DUBs UCHL1 and UCHL5 to alter their activity.","evidence":"Monoclonal antibody development, antibody-peptide crystal structure, MS proteomics, and DUB activity assays","pmids":["34326324"],"confidence":"High","gaps":["Functional consequences of altered DUB activity in cells not fully resolved","Full endogenous substrate set still incomplete"]},{"year":2023,"claim":"Extended the chemistry by identifying UBE2W as a writer of aminoacylated-lysine ubiquitination, a novel PTM linking ubiquitin to the α-amine of aminoacylated lysines and driving rapid protein degradation.","evidence":"Genetic code expansion with unnatural amino acids, mass spectrometry, and cell-based ubiquitination assays","pmids":["36593310"],"confidence":"Medium","gaps":["Novel PTM awaits independent replication","Physiological prevalence and regulation of aminoacylated-lysine ubiquitination unclear"]},{"year":2022,"claim":"Proposed an anti-inflammatory role in which UBE2W attenuates TNFα-induced NF-κB signaling and ameliorates experimental colitis.","evidence":"Dual-luciferase reporter, Western blot, immunofluorescence, and a DSS colitis mouse model","pmids":["35314916"],"confidence":"Low","gaps":["No direct substrate identified for the NF-κB inhibitory effect","Single lab, mechanism not reconciled with N-terminal ubiquitination activity"]},{"year":2026,"claim":"Proposed that UBE2W mediates K63-linked ubiquitination of p53 to regulate its localization and impair DNA repair/apoptosis in pancreatic cancer.","evidence":"In vitro/in vivo functional and ubiquitination assays, subcellular fractionation, and knockdown/overexpression","pmids":["41657785"],"confidence":"Low","gaps":["K63-linked chain-building activity conflicts with established N-terminal monoubiquitination mechanism and needs independent verification","Direct p53 modification site not defined","Limited methodological detail"]},{"year":null,"claim":"It remains unresolved how UBE2W's distinct chemistries (α-amino N-terminal monoubiquitination versus the reported lysine-directed and aminoacylated-lysine modifications) are selected in vivo, and which endogenous N-terminally ubiquitinated substrates account for its developmental and tissue-specific phenotypes.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying model reconciling N-terminal versus internal-lysine targeting","Cognate E3 partners and chain-elongating E2s not mapped for most substrates","Physiological substrate set driving knockout phenotypes incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,4,5,10]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,2]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,4]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5]}],"complexes":[],"partners":["STUB1","ATXN3","TRIM5","UBE2N","UBE2V2","FANCL","UCHL1","UCHL5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96B02","full_name":"Ubiquitin-conjugating enzyme E2 W","aliases":["E2 ubiquitin-conjugating enzyme W","N-terminal E2 ubiquitin-conjugating enzyme","N-terminus-conjugating E2","Ubiquitin carrier protein W","Ubiquitin-conjugating enzyme 16","UBC-16","Ubiquitin-protein ligase W"],"length_aa":151,"mass_kda":17.3,"function":"Accepts ubiquitin from the E1 complex and catalyzes its covalent attachment to other proteins (PubMed:20061386, PubMed:21229326). Specifically monoubiquitinates the N-terminus of various substrates, including ATXN3, MAPT/TAU, POLR2H/RPB8 and STUB1/CHIP, by recognizing backbone atoms of disordered N-termini (PubMed:23560854, PubMed:23696636, PubMed:25436519). Involved in degradation of misfolded chaperone substrates by mediating monoubiquitination of STUB1/CHIP, leading to recruitment of ATXN3 to monoubiquitinated STUB1/CHIP, and restriction of the length of ubiquitin chain attached to STUB1/CHIP substrates by ATXN3. After UV irradiation, but not after mitomycin-C (MMC) treatment, acts as a specific E2 ubiquitin-conjugating enzyme for the Fanconi anemia complex by associating with E3 ubiquitin-protein ligase FANCL and catalyzing monoubiquitination of FANCD2, a key step in the DNA damage pathway (PubMed:19111657, PubMed:21229326). In vitro catalyzes 'Lys-11'-linked polyubiquitination. UBE2W-catalyzed ubiquitination also occurs in the presence of inactive RING/U-box type E3s, i.e. lacking the active site cysteine residues to form thioester bonds with ubiquitin, or even in the absence of E3, albeit at a slower rate (PubMed:25436519)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q96B02/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBE2W","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":383,"dependency_fraction":0.028720626631853787},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/UBE2W","total_profiled":1310},"omim":[{"mim_id":"614277","title":"UBIQUITIN-CONJUGATING ENZYME E2 W; UBE2W","url":"https://www.omim.org/entry/614277"},{"mim_id":"613984","title":"FANCD2 GENE; FANCD2","url":"https://www.omim.org/entry/613984"},{"mim_id":"608111","title":"FANCL GENE; FANCL","url":"https://www.omim.org/entry/608111"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBE2W"},"hgnc":{"alias_symbol":["FLJ11011","UBC-16"],"prev_symbol":[]},"alphafold":{"accession":"Q96B02","domains":[{"cath_id":"3.10.110.10","chopping":"1-139","consensus_level":"medium","plddt":90.2578,"start":1,"end":139}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96B02","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96B02-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96B02-F1-predicted_aligned_error_v6.png","plddt_mean":87.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBE2W","jax_strain_url":"https://www.jax.org/strain/search?query=UBE2W"},"sequence":{"accession":"Q96B02","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96B02.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96B02/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96B02"}},"corpus_meta":[{"pmid":"21855799","id":"PMC_21855799","title":"Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP.","date":"2011","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/21855799","citation_count":143,"is_preprint":false},{"pmid":"23560854","id":"PMC_23560854","title":"Ube2W conjugates ubiquitin to α-amino groups of protein N-termini.","date":"2013","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/23560854","citation_count":90,"is_preprint":false},{"pmid":"26101372","id":"PMC_26101372","title":"TRIM5α requires Ube2W to anchor Lys63-linked ubiquitin chains and restrict reverse transcription.","date":"2015","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/26101372","citation_count":82,"is_preprint":false},{"pmid":"23696636","id":"PMC_23696636","title":"The ubiquitin-conjugating enzyme (E2) Ube2w ubiquitinates the N terminus of substrates.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23696636","citation_count":82,"is_preprint":false},{"pmid":"25436519","id":"PMC_25436519","title":"Intrinsic disorder drives N-terminal ubiquitination by Ube2w.","date":"2014","source":"Nature chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/25436519","citation_count":63,"is_preprint":false},{"pmid":"36593310","id":"PMC_36593310","title":"Genetic code expansion reveals aminoacylated lysine ubiquitination mediated by UBE2W.","date":"2023","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/36593310","citation_count":27,"is_preprint":false},{"pmid":"21229326","id":"PMC_21229326","title":"UBE2W interacts with FANCL and regulates the monoubiquitination of Fanconi anemia protein FANCD2.","date":"2010","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/21229326","citation_count":26,"is_preprint":false},{"pmid":"26601958","id":"PMC_26601958","title":"Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26601958","citation_count":23,"is_preprint":false},{"pmid":"28986324","id":"PMC_28986324","title":"The ubiquitin conjugating enzyme Ube2W regulates solubility of the Huntington's disease protein, huntingtin.","date":"2017","source":"Neurobiology of disease","url":"https://pubmed.ncbi.nlm.nih.gov/28986324","citation_count":22,"is_preprint":false},{"pmid":"34326324","id":"PMC_34326324","title":"Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34326324","citation_count":19,"is_preprint":false},{"pmid":"23709311","id":"PMC_23709311","title":"Biochemical and structural characterization of the ubiquitin-conjugating enzyme UBE2W reveals the formation of a noncovalent homodimer.","date":"2013","source":"Cell biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/23709311","citation_count":14,"is_preprint":false},{"pmid":"27185577","id":"PMC_27185577","title":"Loss of ubiquitin E2 Ube2w rescues hypersensitivity of Rnf4 mutant cells to DNA damage.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27185577","citation_count":13,"is_preprint":false},{"pmid":"31710394","id":"PMC_31710394","title":"UBE2W down-regulation promotes cell apoptosis and correlates with hypospermatogenesis.","date":"2019","source":"Andrologia","url":"https://pubmed.ncbi.nlm.nih.gov/31710394","citation_count":7,"is_preprint":false},{"pmid":"26525192","id":"PMC_26525192","title":"Biochemical and structural characterization of a novel ubiquitin-conjugating enzyme E2 from Agrocybe aegeria reveals Ube2w family-specific properties.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/26525192","citation_count":6,"is_preprint":false},{"pmid":"35314916","id":"PMC_35314916","title":"UBE2W Improves the Experimental Colitis by Inhibiting the NF-κB Signaling Pathway.","date":"2022","source":"Digestive diseases and sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35314916","citation_count":3,"is_preprint":false},{"pmid":"18616160","id":"PMC_18616160","title":"[Generation of mouse UBE2W antibody and analysis of UBE2W expression in mouse tissues].","date":"2008","source":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/18616160","citation_count":3,"is_preprint":false},{"pmid":"38189387","id":"PMC_38189387","title":"[UBE2W overexpression promotes proliferation of intestinal mucosal cells in mice with chemically induced colitis].","date":"2023","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/38189387","citation_count":0,"is_preprint":false},{"pmid":"41657785","id":"PMC_41657785","title":"UBE2W promotes pancreatic cancer progression through regulating K63-linked ubiquitination of p53.","date":"2026","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/41657785","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.22.676137","title":"Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W","date":"2025-09-22","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.22.676137","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10534,"output_tokens":3503,"usd":0.042073,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11096,"output_tokens":4211,"usd":0.080378,"stage2_stop_reason":"end_turn"},"total_usd":0.122451,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"Ube2w acts as an initiator E2 that monoubiquitinates CHIP (the E3 ligase), and this monoubiquitination stabilizes the interaction between CHIP and the deubiquitinase ataxin-3; ataxin-3 then limits polyubiquitin chain length on CHIP substrates and subsequently deubiquitinates CHIP to terminate the ubiquitination cycle.\",\n      \"method\": \"In vitro ubiquitination assays, co-immunoprecipitation, and functional DUB activity assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and in vitro biochemical assays with multiple orthogonal methods; replicated across functional readouts in a single rigorous study\",\n      \"pmids\": [\"21855799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity; it conjugates ubiquitin to the α-amino group of protein N-termini (including its own N-terminus and that of SUMO) rather than to lysine ε-amino groups.\",\n      \"method\": \"In vitro ubiquitylation assays, mass spectrometry identification of modification site, and biochemical assays with RNF4\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mass spectrometry site identification, independently replicated in same year by Scaglione et al. (PMID:23696636)\",\n      \"pmids\": [\"23560854\", \"23696636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Ube2w ubiquitinates the N-terminal α-NH2 group of substrates (not internal lysines); this was demonstrated using lysine-less and N-terminally blocked versions of ataxin-3 and Tau, and confirmed by direct N-terminal sequencing of ubiquitinated ataxin-3; Ube2w contains novel active-site residues important for this activity.\",\n      \"method\": \"Mass spectrometry, in vitro ubiquitination assays with lysine-less and N-terminally blocked substrates, N-terminal sequencing, active-site mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (MS, mutagenesis, N-terminal sequencing, blocked substrate controls) in a single rigorous study\",\n      \"pmids\": [\"23696636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"UBE2W forms a noncovalent homodimer via residues in the β-sheet region and Loops 4 and 7 of the catalytic domain; mutation of two catalytic domain residues disrupts dimerization, but dimerization is not required for ubiquitin transfer activity; the C-terminal region of UBE2W adopts a noncanonical position distinct from all other known E2 structures.\",\n      \"method\": \"Biochemical dimerization assays, mutagenesis, structural characterization\",\n      \"journal\": \"Cell biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis and biochemical assays in a single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"23709311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Ube2w recognizes the backbone atoms of intrinsically disordered N-termini of substrates (not sequence-specific); its own C-terminus is partly disordered and flexible, and this flexibility is required for substrate binding and modification — point mutations in or removal of the flexible C-terminus inhibits substrate binding and N-terminal ubiquitination.\",\n      \"method\": \"NMR solution ensemble of full-length Ube2w, mutagenesis, in vitro ubiquitination assays, computational modeling\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with functional mutagenesis validation and in vitro reconstitution in a single rigorous study\",\n      \"pmids\": [\"25436519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRIM5α employs Ube2W to anchor Lys63-linked polyubiquitin chains via Ube2W-catalyzed monoubiquitination of TRIM5α (targeting internal lysines 45 and 50, not the N-terminus which is αN-acetylated in cells); these anchored chains are then elongated by Ube2N/Ube2V2; this anchored K63-linked polyUb is required for TRIM5α-mediated restriction of viral reverse transcription.\",\n      \"method\": \"In vitro ubiquitination assays, cell-based E2 depletion, ubiquitin mutant studies, retroviral restriction assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro reconstitution combined with cell-based depletion and functional antiviral readout, multiple orthogonal methods\",\n      \"pmids\": [\"26101372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"UBE2W interacts with the FANCL E3 ligase via FANCL's PHD domain, catalyzes monoubiquitination of FANCL's PHD domain in vitro, and promotes monoubiquitination of FANCD2 in cells in response to UV irradiation (but not MMC), indicating a DNA-damage pathway-specific regulatory role distinct from UBE2T/HHR6.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, overexpression and knockdown in mammalian cells, domain mapping\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assay and in vitro ubiquitination with cellular knockdown/overexpression, single lab\",\n      \"pmids\": [\"21229326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ube2w KO mice show postnatal lethality and defects in testis, thymus, and skin; proteomic analysis of presymptomatic testis shows preferential accumulation of intrinsically disordered proteins in the absence of UBE2W, supporting the model that UBE2W preferentially targets disordered polypeptides in vivo; UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm.\",\n      \"method\": \"Germline knockout mouse generation, proteomics/mass spectrometry, immunofluorescence for subcellular localization, histology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean germline KO with multiple phenotypic readouts, proteomics, and localization by direct imaging\",\n      \"pmids\": [\"26601958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Genetic epistasis in chicken and human cells shows that Ube2w does not have an essential role in the DNA damage response, but that loss of Ube2w suppresses the DNA damage hypersensitivity of Rnf4-deficient cells, indicating that Ube2w and Rnf4 function in distinct pathways in response to DNA damage despite their functional interaction in vitro.\",\n      \"method\": \"Genetic epistasis (double-mutant/knockdown), DNA damage sensitivity assays in chicken and human cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in two cell systems, single lab\",\n      \"pmids\": [\"27185577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ube2w deficiency markedly decreases mutant huntingtin (mHTT) aggregate formation and increases soluble monomeric mHTT in cultured cells and in HdhQ200 knock-in mice, while reducing mHTT-induced cytotoxicity; this places Ube2w upstream of mHTT aggregation and implicates N-terminal ubiquitination of the disordered HTT N-terminus in promoting aggregation.\",\n      \"method\": \"Ube2w knockdown/KO in immortalized cells, primary neurons, and knock-in mouse model; filter trap aggregation assays; cell viability assays\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in multiple model systems with specific aggregation and viability readouts, single lab\",\n      \"pmids\": [\"28986324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBE2W N-terminally ubiquitinates UCHL1 and UCHL5 at their N-termini; N-terminal ubiquitination of these deubiquitinases distinctly alters their DUB activity; antibodies selectively recognizing N-terminal diglycine remnants (not isopeptide-linked diglycine on lysine) were used to map endogenous UBE2W substrates by mass spectrometry proteomics.\",\n      \"method\": \"Monoclonal antibody development, antibody-crystal structure determination, mass spectrometry proteomics, DUB activity assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of antibody-peptide complex, MS proteomics for substrate mapping, and functional DUB activity assays in a single study\",\n      \"pmids\": [\"34326324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UBE2W acts as a writer of aminoacylated lysine ubiquitination, a newly identified PTM in which ubiquitin is attached to the α-amine of aminoacylated lysine residues; over 2,000 such ubiquitination sites were identified on all 20 aminoacylated lysines in human cell lines, and this modification mediates rapid protein degradation.\",\n      \"method\": \"Genetic code expansion with unnatural amino acids as chemical probes, mass spectrometry, cell-based ubiquitination assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic code expansion strategy with MS validation, single lab, novel PTM requiring independent replication\",\n      \"pmids\": [\"36593310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBE2W inhibits TNFα-induced NF-κB transcriptional activity by attenuating IκB and p65 phosphorylation and blocking nuclear entry of p65; UBE2W overexpression ameliorates DSS-induced colitis in mice with reduced pro-inflammatory cytokine expression.\",\n      \"method\": \"Dual-luciferase reporter assay, Western blot, immunofluorescence, mouse colitis model with UBE2W overexpression/knockdown\",\n      \"journal\": \"Digestive diseases and sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, cell-based reporter and mouse model, no direct mechanistic substrate identified for NF-κB pathway inhibition\",\n      \"pmids\": [\"35314916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"UBE2W mediates K63-linked ubiquitination of p53, regulates p53 subcellular localization, and thereby impairs p53-mediated DNA damage repair and apoptotic pathways in pancreatic cancer cells.\",\n      \"method\": \"In vitro and in vivo functional experiments, ubiquitination assays, subcellular fractionation, knockdown/overexpression\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, limited methodological detail in abstract, mechanistic claim of K63-linked ubiquitination conflicts with established N-terminal ubiquitination activity of UBE2W and requires independent verification\",\n      \"pmids\": [\"41657785\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBE2W is an E2 ubiquitin-conjugating enzyme that uniquely catalyzes monoubiquitination at the α-amino group of protein N-termini, preferentially targeting substrates with intrinsically disordered N-termini by recognizing backbone atoms rather than sequence; this activity is mediated by its own flexible, partly disordered C-terminus, and it functions in diverse cellular processes including initiating CHIP ubiquitination cycles (with ataxin-3 as a regulatory DUB partner), anchoring Lys63-linked polyubiquitin chains on TRIM5α for antiviral restriction, promoting huntingtin aggregation, and regulating FANCD2 monoubiquitination in the DNA damage response.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"UBE2W is an E2 ubiquitin-conjugating enzyme that catalyzes a distinctive form of monoubiquitination, conjugating ubiquitin to the α-amino group of protein N-termini rather than to the ε-amino group of internal lysines [#1, #2]. Substrate selection is structural rather than sequence-specific: UBE2W recognizes the backbone atoms of intrinsically disordered N-termini, and its own flexible, partly disordered C-terminus is required for substrate engagement and N-terminal modification [#4]. Consistent with this preference, loss of UBE2W in vivo causes preferential accumulation of intrinsically disordered proteins [#7]. Through this activity UBE2W functions as an initiator E2 across several pathways: it monoubiquitinates the E3 ligase CHIP to nucleate a ubiquitination cycle regulated by the deubiquitinase ataxin-3 [#0], anchors Lys63-linked polyubiquitin chains on TRIM5α (via internal lysines K45/K50) that are then elongated by Ube2N/Ube2V2 to drive antiviral restriction [#5], and N-terminally ubiquitinates the deubiquitinases UCHL1 and UCHL5 to alter their catalytic activity [#10]. UBE2W also promotes mutant huntingtin aggregation by acting upstream of aggregate formation [#9] and contributes to regulation of FANCD2 monoubiquitination in the UV-damage response [#6]. Germline knockout mice display postnatal lethality with defects in testis, thymus, and skin, and UBE2W is a ubiquitously expressed cytoplasmic enzyme [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Before its catalytic specificity was known, UBE2W was placed in a DNA-damage context by showing it engages the FANCL E3 ligase and modulates FANCD2 monoubiquitination, establishing a pathway-specific role distinct from the canonical UBE2T E2.\",\n      \"evidence\": \"Co-IP, domain mapping, in vitro ubiquitination and cellular knockdown/overexpression in mammalian cells\",\n      \"pmids\": [\"21229326\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define the chemical nature of the ubiquitin linkage formed\", \"UV-specific but not MMC-specific role left the in vivo relevance unresolved\", \"No structural basis for FANCL recognition\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established UBE2W as an initiator E2 that monoubiquitinates the E3 ligase CHIP, defining a regulatory ubiquitination cycle terminated by the DUB ataxin-3.\",\n      \"evidence\": \"In vitro ubiquitination, reciprocal co-IP, and DUB activity assays\",\n      \"pmids\": [\"21855799\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Site of CHIP modification not chemically defined at this stage\", \"In vivo significance of the cycle not tested\", \"Did not establish substrate-selection principles\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the central mechanistic question of UBE2W chemistry by demonstrating it conjugates ubiquitin to the α-amino group of protein N-termini, not internal lysines, redefining it as a protein N-terminal monoubiquitylation enzyme.\",\n      \"evidence\": \"In vitro ubiquitylation with lysine-less and N-terminally blocked substrates, mass spectrometry, N-terminal sequencing, and active-site mutagenesis\",\n      \"pmids\": [\"23560854\", \"23696636\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not explain how disordered substrates are recognized\", \"Physiological substrate repertoire unknown\", \"Structural basis of N-terminal targeting unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Characterized UBE2W as a noncovalent homodimer with a noncanonical C-terminal position distinct from other E2s, showing dimerization is dispensable for ubiquitin transfer.\",\n      \"evidence\": \"Biochemical dimerization assays, mutagenesis, and structural characterization\",\n      \"pmids\": [\"23709311\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional purpose of dimerization unclear\", \"Did not link the noncanonical C-terminus to substrate recognition\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Explained substrate selectivity by showing UBE2W recognizes backbone atoms of intrinsically disordered N-termini and that its own flexible C-terminus is required for substrate binding and modification.\",\n      \"evidence\": \"NMR solution ensemble of full-length Ube2w, mutagenesis, in vitro ubiquitination, and computational modeling\",\n      \"pmids\": [\"25436519\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not enumerate endogenous disordered substrates\", \"Affinity determinants for specific substrates not mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated a defined antiviral function in which UBE2W monoubiquitinates internal lysines of TRIM5α to anchor K63 chains elongated by Ube2N/Ube2V2, required for restriction of viral reverse transcription.\",\n      \"evidence\": \"In vitro ubiquitination, cell-based E2 depletion, ubiquitin mutant studies, and retroviral restriction assays\",\n      \"pmids\": [\"26101372\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"TRIM5α is targeted at internal lysines (N-terminus α-acetylated), showing context-dependent site selection not fully generalized\", \"Recruitment mechanism to TRIM5α not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established the in vivo requirement for UBE2W and validated its disordered-protein preference through a knockout mouse showing postnatal lethality and preferential accumulation of intrinsically disordered proteins.\",\n      \"evidence\": \"Germline knockout mice, proteomics, immunofluorescence localization, and histology\",\n      \"pmids\": [\"26601958\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific substrate drivers of phenotypes not identified\", \"Did not connect organismal phenotypes to specific N-terminal ubiquitination events\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Genetic epistasis clarified that despite an in vitro interaction with RNF4, UBE2W and RNF4 act in distinct DNA-damage pathways, and UBE2W is not essential for the DNA damage response.\",\n      \"evidence\": \"Genetic epistasis with DNA damage sensitivity assays in chicken and human cells\",\n      \"pmids\": [\"27185577\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which Ube2w loss suppresses Rnf4-deficient hypersensitivity unknown\", \"Relevant substrates in the pathway not identified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked UBE2W to neurodegeneration by placing it upstream of mutant huntingtin aggregation, implicating N-terminal ubiquitination of the disordered HTT N-terminus in promoting aggregate formation and toxicity.\",\n      \"evidence\": \"Knockdown/KO in cells, primary neurons, and HdhQ200 knock-in mice; filter trap and viability assays\",\n      \"pmids\": [\"28986324\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct N-terminal ubiquitination of HTT not biochemically demonstrated in this study\", \"Whether effect generalizes to other aggregation-prone disordered proteins not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Enabled endogenous substrate mapping with N-terminal-diGly-specific antibodies and showed UBE2W N-terminally ubiquitinates the DUBs UCHL1 and UCHL5 to alter their activity.\",\n      \"evidence\": \"Monoclonal antibody development, antibody-peptide crystal structure, MS proteomics, and DUB activity assays\",\n      \"pmids\": [\"34326324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences of altered DUB activity in cells not fully resolved\", \"Full endogenous substrate set still incomplete\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended the chemistry by identifying UBE2W as a writer of aminoacylated-lysine ubiquitination, a novel PTM linking ubiquitin to the α-amine of aminoacylated lysines and driving rapid protein degradation.\",\n      \"evidence\": \"Genetic code expansion with unnatural amino acids, mass spectrometry, and cell-based ubiquitination assays\",\n      \"pmids\": [\"36593310\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Novel PTM awaits independent replication\", \"Physiological prevalence and regulation of aminoacylated-lysine ubiquitination unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Proposed an anti-inflammatory role in which UBE2W attenuates TNFα-induced NF-κB signaling and ameliorates experimental colitis.\",\n      \"evidence\": \"Dual-luciferase reporter, Western blot, immunofluorescence, and a DSS colitis mouse model\",\n      \"pmids\": [\"35314916\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct substrate identified for the NF-κB inhibitory effect\", \"Single lab, mechanism not reconciled with N-terminal ubiquitination activity\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Proposed that UBE2W mediates K63-linked ubiquitination of p53 to regulate its localization and impair DNA repair/apoptosis in pancreatic cancer.\",\n      \"evidence\": \"In vitro/in vivo functional and ubiquitination assays, subcellular fractionation, and knockdown/overexpression\",\n      \"pmids\": [\"41657785\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"K63-linked chain-building activity conflicts with established N-terminal monoubiquitination mechanism and needs independent verification\", \"Direct p53 modification site not defined\", \"Limited methodological detail\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how UBE2W's distinct chemistries (α-amino N-terminal monoubiquitination versus the reported lysine-directed and aminoacylated-lysine modifications) are selected in vivo, and which endogenous N-terminally ubiquitinated substrates account for its developmental and tissue-specific phenotypes.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying model reconciling N-terminal versus internal-lysine targeting\", \"Cognate E3 partners and chain-elongating E2s not mapped for most substrates\", \"Physiological substrate set driving knockout phenotypes incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 4, 5, 10]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 4]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"STUB1\",\n      \"ATXN3\",\n      \"TRIM5\",\n      \"UBE2N\",\n      \"UBE2V2\",\n      \"FANCL\",\n      \"UCHL1\",\n      \"UCHL5\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}